Probing the Selectivity and Protein⋅Protein Interactions of a Nonreducing Fungal Polyketide Synthase Using Mechanism-Based Crosslinkers

• An activity-based crosslinker successfully detects PKS interdomain interactions
• The crosslinking efficiency is correlated with starter unit specificity of KSs
• The ACPs and KSs from NR-PKSs are interchangeable for crosslinking
• Mutations identify KS surface residues important for ACP⋅KS interactions

SummaryProtein⋅protein interactions, which often involve interactions among an acyl carrier protein (ACP) and ACP partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal nonreducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ mechanism-based crosslinkers to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP⋅KS interactions and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediates key interactions with the negatively charged ACP surface. Such complementary/matching contact pairs can serve as “adapter surfaces” for future efforts to generate new polyketides using NR-PKSs.

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